An emerging development in proteomics has provided great opportunities for biomarker discovery, drug development, and the like. Currently LC-MS has become one of the major platforms for protein identification. To date the development of new LC-MS technology is widely applied to comprehensive proteomic studies. In combination with protein fractionation, isotopic labeling and LC-MS, large amounts of protein can be identified and quantified in a short period of time. In LC-MS high resolution and high sensitivity LC separation is the determining factor for comprehensive qualitative and quantitative proteomics studies. The fused-silica capillary LC column is an essential component in the LC-MS based proteomic approach since it can provide high resolution separation of peptides with high sensitivity.
A large variety of commercial stationary phases are employed in capillary-based LC-MS systems for proteomics applications. To retain the stationary phase inside the column, tapered tip and fit designs are commonly used. In tapered tip designs, one end of the column is tapered to have 10˜20 um inside diameter to provide a “keystone” effect to retain particles. The fabrication of the tapered tip capillary is simple and has minimized the post-column dead volume, thus reducing the effect of band broadening due to the longitudinal diffusion. However, once the tip is broken or contaminated, after a period of time the whole column has to be replaced with a new one to maintain the stable and sensitive ionization efficiency. In addition, the tapered tip cannot be applied to the fabrication of trapping column.
Columns containing frits to retain packings have significant benefit for both packed analytical and trap columns. In frit designs retaining frits are readily formed and fixed to the walls of a fused silica capillary by the sol-gel reaction or the sinter of a small area of silica gel or porous silica-based sorbents. A packed column with a frit at one end can be used over and over for a long time and may be re-used with new packing materials.
Trap columns are important in the LC-MS system. To concentrate and desalt a sample for higher sensitivity analysis, a trap column is often used upstream of an analytical column in nano-LC-MS systems. Unfortunately, the trap column can often be blocked by un-removed gel species or particles from sample solutions after batch analyses or it can lose the concentration factor after numerous sample analyses. Hence trap columns for nano-LC systems need to be changed frequently to sustain the original performance, and, therefore, trap columns can become an expensive cost item in a proteomics lab that routinely runs protein samples. To manufacture the trap columns or analytical columns, frit preparation methods are often the key point for the success of packed columns.
To date, many methods have been provided to improve the frit preparation. Unfortunately, the frit fabrication is not highly reproducible, leading to variations in column performance. In addition, capillaries are fragile at the position of the frit since the polyimide coating is often removed during frit fabrication (http://parteq.technologypublisher.com/technology/4510 Multi-Channel Optical Fiber as a Frit.). Recently, tapered tips or tapered tips with sintered fits (Proteomics 2010, 10, 1-4: A simple and efficient frit preparation method for one end tapered-fused silica-packed capillary columns in nano-LC-ESI MS) have been used instead of conventional frits to retain the stationary phase in the column; and also directly used as an electro-sprayer in LC-MS. However, the frequent clogging, breaking and contamination of the tip can cause the replacement of the entire packed column and therefore limits the use of the packed column with tapered tip. Moreover, because of the fragile nature and unfit size for the fitting, the tapered packed columns are not adequate to be used as trap columns.
To retain the polyimide coating and be adequate for trap columns, various sol-gel techniques for on-column fit preparation have been reported (J. High Resol Chro, 1999, 22, 438: AC 2003, 75, 4292). More recently, by dipping the sol-gel solution to a capillary column with dry Si resins inside, a simple low backpressure and inexpensive on-column fit fabrication method has been developed. However, the frit thus formed can often be flushed out during the application of high pressure. An increase of the frit strength by increasing the capillary action time of the sol-gel solution in the packed end can results in extremely high backpressure of frits which is not suitable for high pressure LC applications.
In nano-LC-MS or nanoUPLC-MS systems, because of the use of 3 μm or 1.7 μm 100 Å particles, the resulting back-pressure can be extremely high and range from 5000 to 9000 psi. To manufacture the trap column for such high pressure systems, the trap column is crucial to have certain characteristics. First, because of ultra high pressure supply, the polyimide coating on the column end has to be maintained to prevent the column from breaking. In addition, the frit needs high mechanical strength for ultra high pressure resistance. Moreover, low back pressure of the frit can prevent additional pressure which may result in overpressure of the pumps during analysis.